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chitosan solid dispersion: Preparation, characterization and cytotoxicity evaluation
Accepted Manuscript A novel tanshinone IIA/chitosan solid dispersion: Preparation, characterization and cytotoxicity evaluation Chao Luo, Weibin Wu, Xinyu Lin, Yaoqi Li, Kai Yang PII:
S1773-2247(18)31211-5
DOI:
https://doi.org/10.1016/j.jddst.2018.11.024
Reference:
JDDST 846
To appear in:
Journal of Drug Delivery Science and Technology
Received Date: 15 October 2018 Revised Date:
18 November 2018
Accepted Date: 25 November 2018
Please cite this article as: C. Luo, W. Wu, X. Lin, Y. Li, K. Yang, A novel tanshinone IIA/chitosan solid dispersion: Preparation, characterization and cytotoxicity evaluation, Journal of Drug Delivery Science and Technology (2018), doi: https://doi.org/10.1016/j.jddst.2018.11.024. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT A novel tanshinone IIA/chitosan solid dispersion: preparation, characterization and cytotoxicity evaluation Chao Luo a, Weibin Wu b, Xinyu Lin a, Yaoqi Li a, Kai Yang c, * a
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Department of Medicine and Health, Shaoxing University Yuanpei College, Shaoxing, China, 312000; b Department of Basic Medicine, Zhaoqing Medical College, Zhaoqing, China, 526020; c State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, Guangdong Key Laboratory of Vascular Disease, Guangzhou Institute of Respiratory Health, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou, China, 510182.
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Running title: A novel tanshinone IIA/chitosan solid dispersion *
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Address correspondence to: Kai Yang, Ph.D. Associate Professor of Medicine State Key Laboratory of Respiratory Disease Guangzhou Institute of Respiratory Health The First Affiliated Hospital of Guangzhou Medical University 195 Dong Feng Xi Road, Guangzhou, Guangdong, China, 510182 Email: [email protected]
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Author Contributions: C.L. initiated and designed the project, performed the experiments, analyzed data and wrote the manuscript; W.W., X.L., and Y.L. performed the experiments; K.Y. contributed to the design of the project, provided consultation and edited the manuscript. All authors accepted the submission of the manuscript.
ACCEPTED MANUSCRIPT ABSTRACT The objective of this study was to develop and evaluate a novel tanshinone IIA (TA)/chitosan (CS) solid dispersion (TA-CS-SD) to improve the bioavailability. Solid
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dispersion of tanshinone IIA with chitosan was prepared by a pH inversion method via the pH-responsive dissolution property of chitosan matrix. The in vitro dissolution study confirmed dramatically elevated dissolution rate of TA by solid dispersion
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approach, and the release mechanism exhibited pH-responsive performance. The solid
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dispersion was characterized by x-ray diffractometry (XRD), differential scanning calorimetry (DSC), fourier-transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM). Our results showed that TA existed in the form of microcrystal or superfine crystal in solid dispersion, and the reduction of crystallinity
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was correlated with the content of CS matrix. In vitro cytotoxicity study was performed in human non-small cell lung cancer A549 cell line by CCK-8 assay. Significant improvement in antitumor activity of TA in TA-CS-SD was achieved
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compared to the crystalline drug. These results indicated that, compared with free TA,
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TA-CS-SD demonstrated higher dissolution rate and bioactivity. Keywords: Tanshinone IIA, Chitosan, Solid dispersion, pH-responsive, cytotoxicity
ACCEPTED MANUSCRIPT 1. INTRODUCTION Tanshinone IIA (TA), a lipid-soluble bioactive ingredient extracted and purified from the root of Salvia miltiorrhiza Bunge (Danshen) [1], which has been widely used the
treatment
of
cardiovascular
disorders,
cerebrovascular
diseases,
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for
neurodegenerative diseases, pulmonary hypertension and cancer [2-8]. However, the clinical application of TA is hindered by its poor water-solubility (2.8 ng·mL-1) [9],
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short half-life (1-2 h) [10], first pass metabolism [11], and low oral bioavailability.
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Currently, numerous new drug delivery systems have been employed to overcome these issues, such as the nanoparticles [12], polymeric micelles [13], micro-emulsions [14], solid dispersion (SD) [15], intravenous lipid emulsion [16], etc. SD is a highly effective strategy for improving the solubility, dissolution, and
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bioavailability of poorly water-soluble drugs [17]. The mechanisms of increasing drug solubility by SD technology is due to reduced particle size [18], improved wettability [19], increased porosity [20], and alteration in the polymorphic state of drugs from
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crystalline to an amorphous form [21]. Synthetic polymer materials, such as
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polyethylene glycols (PEG) [22], polyvinylpyrrolidone (PVP) [23], hydroxypropyl methylcellulose (HPMC) [24], poloxamers [25], have been widely used in solid dispersion for improving dissolution characteristics and bioavailability of poorly aqueous-soluble drugs. Nowadays, the application of bio-macromolecules, such as alginate [26], chitosan (CS) [27], and cellulose [28], as the carriers for SD has been widely tested and proved to be ideal vehicles. CS, a natural biopolymer derived from chitin and is the second rich nature
ACCEPTED MANUSCRIPT polysaccharide after cellulose, has been widely used as carriers of drug delivery system due to its favorable chemical and biological properties, such as biocompatibility, biodegradability, pH-responsive, adsorptivity, and its hydrophilic
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and mucoadhesive nature [29-31]. Moreover, based on its excellent characteristics, CS has attracted more and more attention as a carrier for hydrophobic drugs to increase the solubility, dissolution, and bioavailability. The CS-based drug delivery carriers,
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such as microspheres [32], micelles [33] and nanoparticles [34], have been reported as
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supporters for loading hydrophobic drugs, Jain et al. [35] fabricated a docetaxel loaded CS nanoparticle, which represents a 25% increase in cytotoxicity compared with the free drug. And recently, more convenient methods such as SD, direct adsorption of powder are also fabricated for loading hydrophobic drugs on CS [36].
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For example, Crucitti et al. [27] reported an abietic acid/chitosan solid dispersion, which can improve the water solubility of abietic acid and promote its bioactivity. To date, there is no report about using CS as carrier for fabricating the solid
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dispersion of TA. Based on the successful development of CS-SD system and given
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the long history of successful usage of TA in clinical treatment of substantial diseases, here in this study, for the first time, we develop a novel tanshinone IIA/chitosan solid dispersion (TA-CS-SD) by using chitosan as carrier to improve the dissolution rate of TA, which provides sustained release performance and pH-sensibility. It is well known that chitosan is insoluble at neutral or slightly alkaline pH and highly soluble at acidic environment [37], and can be regenerated with pH inversion [38]. So, in this study, a rapid one-pot approach for TA-CS-SD is developed by inverting pH of the
ACCEPTED MANUSCRIPT chitosan acetic acid aqueous dispersed with TA. TA is dissolved in ethyl alcohol, and then mixed with chitosan solution, the viscous polymer solution can effectively disperse and adsorb the TA molecules, after that, with the regeneration of CS, the TA
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is coated into the CS matrix, and homogeneous dispersion. The dissolution rate improvement and sustained release performance of the TA-CS-SD are evaluated. The scanning electron microscopy (SEM) is used to examine the morphological structure
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of the TA-CS-SD. The physical and chemical compatibility between the TA and CS in
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SD are characterized by fourier-transform infrared spectroscopy (FTIR). X-ray diffraction (XRD) and differential scanning calorimeter (DSC) are employed to demonstrate that the crystallization degree of TA is decreased after encapsulated into chitosan matrix. Subsequently, in vitro degradation research and swelling experiment
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are performed to assess the pH-sensitive of the TA-CS-SD. The human non-small cell lung cancer A549 cell line is used for the cytotoxicity test to verify the bioactivity
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improvement of solid dispersion.
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2. MATERIALS AND METHODS 2.1 Materials
Pharmaceutical grade, 95% deacetylated chitosan with a viscosity average
molecular weight of 1.2 × 105 was supplied by Aoxing Biotechnology Co., Ltd. (Zhejiang, China). Tanshinone IIA (98%) was purchased from Hao Xuan biological Co., Ltd. (Xi’an, China). Methanol (HPLC grade) was purchased from Aladdin Industrial Corporation (Shanghai, China). Deionized water was used throughout,
ACCEPTED MANUSCRIPT produced by Milli-Q Reference Water Purification System (Merck Millipore, USA). All other chemicals were purchased from commercial sources and used as received. The human non-small cell lung cancer A549 cell line was purchased from the
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Cell Bank of the Chinese Academy of Sciences (Shanghai, China). RPMI1640 medium and fetal bovine serum (FBS) were supplied by Gibco (Carlsbad, USA). Glutamine was from Sigma-Aldrich Trade Co. (USA). The Cell Counting Kit-8
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(CCK-8) was purchased from Beyotime Biotechnology Co (Shanghai, China).
2.2 Preparation of TA-CS-SD and physical mixture (PM)
TA-CS-SD was prepared using the pH inversion method with TA and CS at different mass ratio (1:5, 1:10, 1:20 and 1:40). Briefly, two solutions were prepared: in
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solution A, certain amounts of CS (0.25, 0.5, 1.0, 1.5, 2.0 g, respectively) were dissolved in 100 ml 1 wt% acetic acid solution under magnetic stirring, and in solution B, 50 mg of TA was dissolved in anhydrous alcohol with a final
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concentration of 1 mg/mL. Then, Solution B was added slowly into solution A with
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stirring at 1000 rpm for 30min. subsequently, the resultant mixture solution was neutralized with 1 wt% NaOH solution, washed to neutral with deionized water, and then freeze-dried. The yield of the obtained TA-CS-SD was calculated by the following formula, and the final products were stored in a desiccator for further experiments. The regenerative CS was prepared following the above methods except for the addition of TA, named CS-SD. The physical mixture (PM) was prepared by mixing TA and CS-SD at the weight ratio of 1:10.
ACCEPTED MANUSCRIPT 1
% =
× 100%
2.3 Analytical methodology
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The concentration of TA in the testing medium were quantified by a Waters e2695-2489 liquid chromatographic system (HPLC) under the following conditions: SunFire C18 (4.6mm ×250 mm, 5 µm) column, methanol and water (90:10 v/v) as
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mobile phase at a flow rate of 1.0 ml/min, UV detection at 270 nm. The linearity of
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the method was investigated in the concentration range of 1.0-20.0 µg/ml (r=0.9996). The RSD of intraday and interday precision for TA were below 2%.
2.4 Encapsulation efficiency and dissolution test in vitro
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A 5.0 mg of TA-CS-SD was dissolved in 10.0 ml of 0.1 M hydrochloric acid solution (pH 1.2) and centrifuged at 12000 rpm for 10 min, and 1.0 ml of the supernatant was diluted to 10.0 ml with anhydrous alcohol. The TA in the solution
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was determined by HPLC at wavelength of 270 nm. The drug loading content and
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encapsulation efficiency were calculated via the following equations: 2 $%& '( )& *')+ )+ % =
3 e)*(34% (+ ') 55 * )*6 % =
,
,-./-/0
,-./-/0 ,
× 100%
,-./-/0
,
× 100%
The in vitro dissolution study was performed using USP type II (paddle type apparatus) at 100 rpm, 37 ± 0.5 °C using 900 ml of simulated intestinal fluid (pH 7.4 phosphate buffer solution contained 0.5% sodium dodecyl sulfate) or simulated
ACCEPTED MANUSCRIPT gastric fluid (0.1 mol/L HCl, pH 1.2) as the dissolution medium. Briefly, TA-CS-SD or PM equivalent to 5.0 mg TA were sealed into dialysis bag and immersed in dissolution medium. At pre-determined time points, 5 mL of the sample was
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withdrawn and the equal volume of dissolution medium was instantly replaced to maintain a constant dissolution volume. The samples were filtered through 0.45 µm millipore filter and analyzed by HPLC as described above. Experiments were carried
2.5 Characterization of TA-CS-SD
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out in triplicate.
2.5.1 Powder X-ray diffraction (XRD)
The XRD was performed on a X'pert PRO X-ray diffraction system (PANalytical,
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Netherlands) with Cu Kα radiation, at a voltage of 40 kV and a current of 40 mA. The scanning rate was 2°/min over a 2θ range of 5–60°.
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2.5.2 Differential scanning calorimetry (DSC)
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The thermal behavior of TA-CS-SD and PM were performed with a Mettler Toledo TGA/DSC 2 STARe system. Samples were crimped in aluminium crucible and heated at a rate of 20 °C/min from 25 °C to 300 °C. Nitrogen was used as the purge gas at a flow rate of 20 mL/min.
2.5.3 Fourier-transform infrared spectroscopy (FTIR) The FTIR analysis was performed with a Nicolet 740 spectrometer (Thermo
ACCEPTED MANUSCRIPT Scientific, USA). The pellets were prepared by crushing samples with potassium bromide (KBr). Scans were obtained at a resolution of 4 cm-1 over a wavenumber
2.5.4 Scanning Electron Microscopy (SEM)
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range of 4000-400 cm-1.
The morphology of TA-CS-SD, PM, TA and CS-SD was observed with a
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JEM-6360 SEM. The samples were mounted on metal stubs using adhesive tape and
SEM.
2.6 Degradation studies
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made electrically conductive by sputter-coating with gold and then observed with
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The in vitro degradation of CS-SD was carried out in 0.1 M HCl solution (pH 1.2). CS-SD sample was dispersed in 10.0 ml of buffer, incubated in a 37 ℃ shaker at 150 r/min. At regular intervals, the samples were placed in the ultra-filtration
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centrifuge tubes (MWCO 50K), centrifuged at 12000 rpm for 15 min, and dried in an
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oven at 60 ℃ to a constant weight. The remaining CS-SD was weighed, and the results were represented as undegraded mass percent versus time. The experiment was performed in triplicate.
2.7 Swelling studies The swelling ratio of CS-SD was carried out in PBS (pH 7.4) at 37 ℃. At regular intervals, the swollen CS-SD was weighed after the excess liquid were carefully
ACCEPTED MANUSCRIPT wiping off with a tissue paper. The swelling ratio (Sw) was calculated by the following equation, where Wd and Ws are the weights of the dried and swollen CS-SD, respectively. Three replicate tests were performed in the swelling studies. % =
9 −9 × 100% 9
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4 8
2.8 Cell viability
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A549 cells were cultured in RPMI1640 medium, supplemented with 10% (V/V)
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FBS and 2 mM glutamine in a humidified incubator at 37°C, 5% CO2 atmosphere. Fresh medium was replaced every 48 h, cells in logarithmic phase will be used in the experiment. TA and TA-CS-SD (1:10) were dispersed in medium at various concentrations (2.5, 5, 10 µM), and TA dissolved in DMSO and diluted by medium at
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the same concentrations were used as comparisons.
Cell proliferation level was determined by CCK-8 assay. Briefly, A549 cells were seeded in 96-well plates at a density of 1×105 cells/0.1ml and cultured for 24 h
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(37 °C, 5% CO2). Then, the medium was removed and cells were treated with
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formulation equivalent to TA at various concentrations (2.5, 5, 10 µM). Medium, DMSO (diluted by medium), and CS-SD (dispersed in medium, 35 µg/ml) were used as control groups, the amount of CS-SD used in cytotoxicity test is equivalent to the mass of CS matrix in TA-CS-SD of high concentration group. The plates were then incubated (37 °C, 5% CO2) for 24 h, and the medium was replaced by 90 µl of basal medium and 10 µl of CCK-8, and continue to incubate for another 4 h. Afterwards, the cell viability was detected by scanning with ELISA plate reader (Thermo
ACCEPTED MANUSCRIPT Scientific™ Multiskan™ FC) at the wavelength of 450 nm. The cell viability was reflected as a percentage of absorbance relative to that of the untreated control. Five
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individual experiments were performed and analyzed in each group.
2.9 Statistical analysis
The results were represented as mean ± standard deviation (mean ± SD).
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Statistical analysis was performed with SPSS 17.0 software. One way analysis of
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variance (ANOVA) and LSD test were used for data analysis. P < 0.05 was considered as statistically significant.
3. RESULTS AND DISCUSSION
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3.1 Drug loading and encapsulation efficiency
Melting method and solvent evaporation method are the two major methods for preparation of SD [39]. The main advantages of these two methods are to offer a
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simple preparation process and can obtain the well yield and encapsulation efficiency.
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However, the melting method requires a high thermal stability of matrix, and the solvent evaporation method needs the well lipophilicity of matrix. Therefore, these methods are not suitable for the preparation of CS-SD. Actually, CS can be regenerated from its acidic solution with pH inversion, and this property can be employed to prepare the CS-SD. In order to evaluate the efficiency of this method for preparing SD, the yield and encapsulation efficiency were firstly studied. The yield, encapsulation efficiency and drug loading of TA-CS-SD with different
ACCEPTED MANUSCRIPT TA and CS mass ratio are shown in Table 1. As seen, the method used in preparation of TA-CS-SD is characterized by high yield and encapsulation efficiency. As the mass ratio of TA and CS increases from 1:5 to 1:10, the encapsulation efficiency of the
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TA-CS-SD increases from 84.71 to 94.55%, and stays at a plateau of 93-94%, likely due to the adsorption capacity of CS. The CS macromolecules in solution can adsorb and disperse the TA, and prevent its crystallization. As the solid dispersion is prepared
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with a higher amount of CS, it has a stronger adsorption and dispersion capacity, and
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represents a higher encapsulation efficiency. However, the drug loading of the TA-CS-SD is decreased from 15.69% to 2.36%, with the mass ratio of TA and CS increase from 1:5 to 1:40, respectively, which is caused by the increase in the
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proportion of the matrix.
3.2 Dissolution behavior of TA-CS-SD
To evaluate the pH-dependent release behavior of the TA-CS-SD, in vitro release
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assay was performed in the simulated intestinal fluid (pH 7.4) and simulated gastric
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fluid (pH 1.2). As is shown in Fig. 1, the release of TA from the solid dispersion represented a quite different release pattern of TA-CS-SD at pH 7.4 and 1.2. In compare with pH 7.4, the drug showed a faster release rate and higher dissolution proportion at pH 1.2, which can be explained by the pH-responsive of CS matrix. CS is highly soluble at acidic environment, but insoluble at neutral or slightly alkaline pH. This hypothesis can be supported by the results of swelling and degradation tests. In the dissolution medium at pH 1.2 (Fig. 1A), the release rate of TA from the solid
ACCEPTED MANUSCRIPT dispersion is decreased consistently with increasing ratio of TA: CS from 1:5 to 1:40 due to the dissolution time of chitosan matrix. PM has a poor dissolution rate in dissolution medium due to the high crystallinity of TA, the cumulative release amount
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in 210 min is below 1%, but the dissolution rate of TA in SD increases significantly, the cumulative release amount within 210 min is up to nearly 100% (except for the group of TA: CS with 1:5). It can be explained that the crystallinity of TA is declined
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following the SD process, and the increased amount of CS matrix may prevent the
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crystallization of TA. These results are further supported by data from XRD and SEM. Whereas at a pH of 7.4 (Fig. 1B), the TA-CS-SD has a well sustained-release performance, with increasing ratio of TA: CS from 1:5 to 1:40, the cumulative release amount at 24 h is decreased from 48% to 32%. The mechanism of TA release from SD
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at pH 7.4 may be predominantly diffusion-controlled, and the swelling property of the CS matrix is the main factor to control the dissolution rate [40]. Collectively, the encapsulation efficiency, drug loading, and dissolution rate are correlated with the
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ratios of drug: polymer in the formulation. Because of the higher encapsulation
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efficiency, drug loading, and dissolution rate, the group of TA-CS-SD (1:10) was selected as a sample used in characterization and in vitro cytotoxicity.
3.3 Characterization
3.3.1 Powder X-ray diffraction (XRD) The XRD patterns of TA, CS, TA-CS-SD, and PM are shown in Fig. 2. The diffraction peaks of CS at around 10° and 20° are characteristic of the hydrated
ACCEPTED MANUSCRIPT crystalline structure [41]. The pure TA shows the characteristically well-defined sharp, narrow diffraction peak of a highly crystalline structure at 2θ of 7.20°, and several weak diffraction peaks at 2θ of 9.51°, 11.88°, 14.42°, and 17.89°. The pattern of PM
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retains the distinctive peaks attributed to the crystalline structure of TA. Compared to PM, the characteristic reflection peak of TA at 2θ 7.20° still remains in the TA-CS-SD, but the relative intensity is lower. This may suggest that TA is still in the form of
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microcrystal in TA-CS-SD, but the crystal growth of TA may be prohibited by CS to
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some extent. Therefore, the crystallinity of TA in TA-CS-SD is relatively lower [15]. Furthermore, with increasing ratio of TA: CS from 1:5 to 1:40, the intensity of the characteristic peak of TA decreased gradually, indicating the reduction of crystallinity is correlated with the content of CS matrix (Fig. 2B). These results can explain the
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high solubility of TA in SD.
3.3.2 Differential scanning calorimetry (DSC)
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As seen in Fig. 3, the CS thermogram shows a wide endothermic band centered
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at approximately 75 °C due to the presence of water. The DSC curve of TA shows a sharp endothermic peak at 219.20 °C corresponding to its melting point, indicating its nature of crystalline state. Because of the high content of CS, it is difficult to discern the endothermic peak of TA in TA-CS-SD and PM [27]. But the endothermic event of TA in TA-CS-SD is altered from 219.20°C to 197.77°C. It may be due to the formation of eutectic mixtures in TA-CS-SD leading to the depression of melting point, and this result also suggests that a reduction of the crystallinity of TA [15],
ACCEPTED MANUSCRIPT which is corresponding to the results of XRD and SEM. The partial reduction of the crystallinity contributes to explaining the increased drug solubility.
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3.3.3 Fourier-transform infrared spectroscopy (FTIR) FTIR spectroscopy was applied to investigate the interactions between TA and CS in solid dispersion. As shown in Fig. 4, CS shows characteristic peaks at 3148
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cm-1 (O-H and N-H stretching vibration), 1650 cm−1 (C=O stretching vibration (amide
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I)), 1578 cm−1 (N-H in-plane bending vibration (amide II)), and 1095 cm−1 (C-O stretching vibration) [42]. For TA, the main characteristic peak is found at 1670 cm-1 (C=O stretching vibration), and the peaks at 2925 and 2866cm-1 are assigned to the C-H stretching vibration of -CH2, and -CH3 [43]. The FTIR spectra of the PM
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comprise a summation of TA and CS. The FTIR spectra of the TA-CS-SD are similar to the spectra of mixture, but many weaker peaks of TA are covered by CS, indicating that TA is uniformly dispersed in the CS matrix, and the quantity of CS is more than
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that of TA, so the signals of CS overshadow many weaker peaks from smaller
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quantities of TA. The results suggest that there are no chemical interactions between TA and carriers.
3.3.4 Morphological analysis The morphology of pure TA, CS, PM, and TA-CS-SD at drug/polymer weight ratios of 1:5, 1:10, and 1:20 were studied via SEM to investigate the potential morphological differences (Fig. 5). The crystal morphology of pure TA is an acicular
ACCEPTED MANUSCRIPT crystal, CS has an irregular shape. Morphology of the PM displayed the crystals of TA and polymer in the mixture. These results confirm the presence of crystalline TA in PM. Conversely, the crystals of TA are invisible in TA-CS-SD at the weight ratios of
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1:10 and 1:20, the TA-CS-SD appears in the form of irregular particles. But, for TA-CS-SD (1:5), the small crystalline TA was observed at the surface of the solid dispersion. These results in combine demonstrated that TA may disperse in solid
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dispersion in microcrystal form, and the crystal growth of TA can be prohibited by CS,
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furthermore, the prohibition effect may be related to the mass ratio of TA and CS. This finding is in accordance with the results of XRD.
3.4 Degradation and Swelling studies
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To elucidate the different release mechanism of SD in different pH, the degradation study and swelling study of CS-SD were conducted in simulated gastric fluid (pH 1.2) and simulated intestinal fluid (pH 7.4). As shown in Fig. 6A, CS-SD is
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almost degraded completely within 90 min, likely due to the well solubility of CS at
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acidic environment. Conversely, CS is insoluble at slightly alkaline pH, but it has well swelling property due to the hydrophilic groups on the CS, the mass of CS-SD increase by about 3-fold within 10 h (Fig. 6B), indicating that TA-CS-SD has different release mechanism in different pH environment, the drug release is related to the degradation of the CS matrix in simulated gastric fluid (pH 1.2), but in simulated intestinal fluid (pH 7.4), the swelling process might be a key factor to control the drug release. These results are in agreement with the previous study [40].
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3.5 Cell viability To evaluate the antitumor activity improvement of TA after it was prepared into
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solid dispersion, the in vitro cytotoxicity was performed using the human non-small cell lung cancer A549 cell line as a cellular model. As is shown in Fig. 7, there is no obvious cell cytotoxicity of the CS-SD group, indicating excellent biocompatibility
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and negligible cytotoxicity of CS matrix. Because of the poor water solubility, the
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group of TA dispersed in medium has little activity on A549. In contrast, the group of TA dissolved in DMSO significantly inhibits the growth of A549 in a dose-dependent manner with a percent decrease of cell viability of 15%, 25%, 37 %, respectively. The cytotoxicity of TA-CS-SD (1:10) is similar to the group of TA dissolved in DMSO at
4. CONCLUSION
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the same concentrations, likely due to the increased solubility of TA in SD.
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In this study, a novel solid dispersion of TA by using CS as a carrier was
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successfully developed. Our data showed that TA-CS-SD prepared by pH inversion method can largely increase the dissolution rate of TA, and the release mechanism of SD exhibited a pH-responsive way. The TA-CS-SD was characterized by XRD, DSC, FTIR, and SEM, with all confirming that TA is present in the form of microcrystal or superfine crystal in SD, and the reduction of crystallinity is correlated with the content of CS matrix. No interactions of drug substances and the carriers are found in SD. Moreover, the antitumor activity of TA, reflected by in vitro cytotoxicity test, is
ACCEPTED MANUSCRIPT significantly enhanced in TA-CS-SD. In conclusion, the SD using CS as a carrier can markedly improve the dissolution rate and bioactivity of TA, and the preparation process of TA-CS-SD, for the first, provides a novel approach to overcoming the low
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dissolution rate and activity of poorly water-soluble drugs, such as TA, and has the potential to largely increase the clinical usage of this group of well-proved clinical
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medication.
ACCEPTED MANUSCRIPT Table 1. The yield, encapsulation efficiency and drug loading of the prepared TA-CS-SD. Encapsulation efficiency
Drug loading
(%)
(%)
(%)
TA-CS-SD(1:5)
90.11±5.05
84.71±2.18
TA-CS-SD(1:10)
97.03±2.96
94.55±3.88
TA-CS-SD(1:20)
97.56±0.88
92.97±4.44
TA-CS-SD(1:40)
97.27±0.40
94.05±3.55
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Yield
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Sample
15.69±0.59 8.86±0.13 4.54±0.21 2.36±0.08
ACCEPTED MANUSCRIPT Declaration of interest The authors disclose no potential conflicts of interest.
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Acknowledgements The authors acknowledge the financial support from the Natural Science Foundation of Zhejiang province (LQ16H310002) and the National Natural Science
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Foundation of China (81800057).
ACCEPTED MANUSCRIPT Reference [1] S. Gao, Z.P. Liu, H. Li, P.J. Little, P.Q. Liu, S.W. Xu, Cardiovascular actions and therapeutic potential of tanshinone IIA, Atherosclerosis. 220 (2012) 3-10.
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[2] J.Y. Han, J.Y. Fan, Y. Horie, S. Miura, D.H. Cui, H. Ishii, T. Hibi, H. Tsuneki, I. Kimura, Ameliorating effects of compounds derived from Salvia miltiorrhiza root
reperfusion, Pharmacol Ther. 117 (2008) 280-295.
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extract on microcirculatory disturbance and target organ injury by ischemia and
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[3] T. Song, Y. Yao, T. Wang, H. Huang, H. Xia, Tanshinone IIA ameliorates apoptosis of myocardiocytes by up-regulation of miR-133 and suppression of Caspase-9, Eur J Pharmacol. 815(2017) 343-350.
[4] X.H. Wang, S.L. Morris-Natschke, K.H. Lee, New developments in the chemistry
133-148.
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and biology of the bioactive constituents of Tanshen, Med Res Rev. 27 (2007)
[5] J.G. Wang, S.C. Bondy, L. Zhou, F.Z. Yang, Z.G. Ding, Y. Hu, Y. Tian, P.Y. Wen,
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H. Luo, F. Wang, W.W. Li, J. Zhou, Protective effect of tanshinone IIA against infarct
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size and increased HMGB1, NFkappaB, GFAP and apoptosis consequent to transient middle cerebral artery occlusion, Neurochem Res. 39(2014) 295-304. [6] X. Liu, M. Ye, C.Y. An, L.Q. Pan, L.T. Ji, The effect of cationic albumin-conjugated PEGylated tanshinone IIA nanoparticles on neuronal signal pathways and neuroprotection in cerebral ischemia, Biomaterials. 34 (2013) 6893-6905. [7] S.C. Chiu, S.Y. Huang, S.P. Chen, C.C. Su, T.L. Chiu, C.Y. Pang, Tanshinone IIA
ACCEPTED MANUSCRIPT inhibits human prostate cancer cells growth by induction of endoplasmic reticulum stress in vitro and in vivo, Prostate Cancer Prostatic Dis. 16(2013) 315-322. [8] C. Lin, L. Wang, H. Wang, L. Yang, H. Guo, X. Wang, Tanshinone IIA inhibits
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breast cancer stem cells growth in vitro and in vivo through attenuation of IL-6/STAT3/NF-κB signaling pathways, J Cell Biochem. 114(2013) 2061-2070.
[9] H.X. Yan, J. Li, Z.H. Li, W.L. Zhang, J.P. Liu, Tanshinone IIA – loaded pellets
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developed for angina chronotherapy: Deconvolution-based formulation design and
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optimization, pharmacokinetic and pharmacodynamic evaluation, Eur J Pharm Sci. 76(2015) 156-164.
[10] W.L. Zhang, H.L. He, J.P. Liu, J. Wang, S.Y. Zhang, S.S. Zhang, Z.M. Wu, Pharmacokinetics and atherosclerotic lesions targeting effects of tanshinone IIA
306-319.
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discoidal and spherical biomimetic high density lipoproteins, Biomaterials. 34(2013)
[11] Y. Zhang, P.X. Jiang, M. Ye, S. H. Kim, C. Jiang, J.X. Lü, Tanshinones: sources,
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pharmacokinetics and anti-cancer activities, Int J Mol Sci. 13(2012) 13621-13666.
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[12] Q. Li, Y. Wang, N. Feng, Z. Fan, J. Sun, Y. Nan, Novel polymeric nanoparticles containing tanshinone IIA for the treatment of hepatoma, J Drug Target. 16(2008) 725-732.
[13] J.M. Zhang, Y.B. Li, X.F. Fang, D.M. Zhou, Y.T. Wang, M.W. Chen, TPGS-g-PLGA/Pluronic F68 mixed micelles for tanshinone IIA delivery in cancer therapy, Int J Pharm. 476 (2014) 185-198. [14] H. Ma, Q. Fan, J. Yu, J. Xin, C. Zhang, Novel microemulsion of tanshinone IIA,
ACCEPTED MANUSCRIPT isolated from Salvia miltiorrhiza Bunge, exerts anticancer activity through inducing apoptosis in hepatoma cells, Am J Chin Med. 41 (2013) 197-210. [15] X.F. Zhai, C.G. Li, G.B. Lenon, C.L. Xue, W.Z. Li, Preparation and
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characterisation of solid dispersions of tanshinone IIA, cryptotanshinone and total tanshinones, Asian J Pharm Sci. 12 (2017) 85-97.
[16] T. Chu, Q. Zhang, H. Li, W.C. Ma, N. Zhang, H. Jin, S.J. Mao, Development of
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activity in vitro, Int J Pharm. 424 (2012) 76-88.
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intravenous lipid emulsion of tanshinone IIA and evaluation of its anti-hepatoma
[17] S. Lee, K. Nam, M.S. Kim, S.W. Jun, J.S. Park, J.S. Woo, S.J. Hwang, Preparation and characterization of solid dispersions of itraconazole by using aerosol solvent extraction system for improvement in drug solubility and bioavailability, Arch
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Pharm Res. 28 (2005) 866-74.
[18] S. Janssens, G. Van den Mooter, Review: physical chemistry of solid dispersions, J Pharm Pharmacol. 61 (2009) 1571-1586.
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[19] N. Zerrouk, C. Chemtob, P. Arnaud, S. Toscani, J. Dugue, In vitro and in vivo
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evaluation of carbamazepine-PEG 6000 solid dispersions, Int. J. Pharm. 225 (2001) 49-62.
[20] J. Li, P. Liu, J.P. Liu, W.L. Zhang, J.K. Yang, Y.Q. Fan, Novel Tanshinone II A ternary solid dispersion pellets prepared by a single-step technique: In vitro and in vivo evaluation, Eur J Pharm Biopharm. 80 (2012) 426-432. [22] D.P. Otto, A. Otto, M. M. Villiers, Experimental and mesoscale computational dynamics studies of the relationship between solubility and release of quercetin from
ACCEPTED MANUSCRIPT PEG solid dispersions, Int. J. Pharm. 456 (2013) 282-292. [23] A. Jahangiri, M. Barzegar-Jalali, A. Garjani, Y. Javadzadeh, H. Hamishehkar, A. Afroozian,
K.
Adibkia,
Pharmacological
and
histological
examination
of
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atorvastatin-PVP K30 solid dispersions, Powder Technol. 286 (2015) 538-545. [24] M.K. Riekes, G. Kuminek, G.S. Rauber, C. Eduardo, M. Campos, A.J. Bortoluzzi, H.K. Stulzer, HPMC as a potential enhancer of nimodipine biopharmaceutical
SC
properties via ball-milled solid dispersions, Carbohyd Polym. 99 (2014) 474-482.
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[25] J.O. Eloy, J.M. Marchetti, Solid dispersions containing ursolic acid in Poloxamer 407 and PEG 6000: A comparative study of fusion and solvent methods, Powder Technol. 253 (2014) 98-106.
[26] J. Guan, Q. Liu, X. Zhang, Y. Zhang, R. Chokshi, H. Wu, S. Mao, Alginate as a
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potential diphase solid dispersion carrier with enhanced drug dissolution and improved storage stability, Eur J Pharm Sci. 114 (2018) 346-355. [27] V.C. Crucitti, L.M. Migneco, A. Piozzi, V. Taresco, M. Garnett, R.H. Argent, I.
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Francolini, Intermolecular interaction and solid state characterization of abietic
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acid/chitosan solid dispersions possessing antimicrobial and antioxidant properties, Eur J Pharm Biopharm. 125 (2018) 114-123. [28] B. Li, S. Konecke, L.A. Wegiel, L.S. Taylor, K.J. Edgar, Both solubility and chemical stability of curcumin are enhanced by solid dispersion in cellulose derivative matrices, Carbohyd Polym. 98 (2013) 1108-1116. [29] M.A. Pujana, L. Pérez-Álvarez, L.C. Iturbe, I. Katime, Biodegradable chitosan nanogels crosslinked with genipin, Carbohyd Polym. 94 (2013) 836-842.
ACCEPTED MANUSCRIPT [30] E. Yilmaz, Z. Yalinca, K. Yahyaa, U. Sirotina, pH responsive graft copolymers of chitosan, Int J Biol Macromol. 90 (2016) 68-74. [31] K. Kim, K. Kim, J.H. Ryu, H. Lee, Chitosan-catechol: A polymer with
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long-lasting mucoadhesive properties, Biomaterials. 52 (2015) 161-170. [32] A.J. Ribeiro, R.J. Neufeld, P. Arnaud, J.C. Chaumeil, Microencapsulation of lipophilic drugs in chitosan-coated alginate microspheres, Int. J. Pharm. 187 (1999)
SC
115-123.
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[33] J.P. Nam, S.C. Park, T.H. Kim, J.Y. Jang, C. Choi, M.K. Jang, J.W. Nah, Encapsulation of paclitaxel into lauric acid-O-carboxymethyl chitosan-transferrin micelles for hydrophobic drug delivery and site-specific targeted delivery, Int. J. Pharm. 457 (2013) 124-135.
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[34] L.H.Chuah, C.J. Roberts, N. Billa, S. Abdullah, R. Rosli, Cellular uptake and anticancer effects of mucoadhesive curcumin-containing chitosan nanoparticles, Colloids Surf B. 116 (2014) 228-236.
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[35] A. Jain, K. Thakur, G. Sharma, P. Kush, U.K. Jain, Fabrication, characterization
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and cytotoxicity studies of ionically cross-linked docetaxel loaded chitosan nanoparticles, Carbohydr Polym. 137 (2016) 65-74. [36] D. Liudvinaviciute, K. Almonaityte, R. Rutkaite, J. Bendoraitiene, R. Klimaviciute, Adsorption of rosmarinic acid from aqueous solution on chitosan powder, Int J Biol Macromol. 118 (2018) 1013-1020. [37] S. Abbad, Z. Zhang, A.Y. Waddad, W.L. Munyendo, H. Lv, J. Zhou, Chitosan-modified cationic amino acid nanoparticles as a novel oral delivery system
ACCEPTED MANUSCRIPT for insulin, J Biomed Nanotechnol. 11 (2015) 486-499. [38] S. Bi, Z. Bao, X. Bai, S. Hu, X. Cheng, X. Chen, Tough chitosan hydrogel based on purified regeneration and alkaline solvent as biomaterials for tissue engineering
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applications, Int J Biol Macromol. 104 (2017) 224-231. [39] C.L. Vo, C. Park, B.J. Lee, Current trends and future perspectives of solid dispersions containing poorly water-soluble drugs, Eur J Pharm Biopharm. 85 (2013)
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799-813.
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[40] S. Hua, H. Yang, W. Wang, A. Wang, Controlled release of ofloxacin from chitosan–montmorillonite hydrogel, Appl Clay Sci. 50 (2010) 112-117. [41] A.F. Martins, D.M. de Oliveira, A.G.B. Pereira, A.F. Rubira, E.C. Muniz, Chitosan/TPP microparticles obtained by microemulsion method applied in controlled
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release of heparin, Int J Biol Macromol. 51 (2012) 1127-1133. [42] B.R. dos Santos, F.B. Bacalhau, T.S. Pereira, C.F. Souza, R. Faez, Chitosan-Montmorillonite microspheres: A sustainable fertilizer delivery system,
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Carbohydr Polym. 127 (2015) 340-346.
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[43] Z. Meng, L. Meng, K. Wang, J. Li, X. Cao, J. Wu, Y. Hu, Enhanced hepatic targeting, biodistribution and antifibrotic efficacy of tanshinone IIA loaded globin nanoparticles, Eur J Pharm Sci. 73 (2015) 35-43.
ACCEPTED MANUSCRIPT Captions 1. Figure 1. Dissolution behavior of TA-CS-SD at different drug: carrier ratios in different medium; (A) simulated gastric fluid (pH1.2) and (B) simulated intestinal
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fluid (pH7.4). 2. Figure 2. XRD patterns of (A) TA, CS-SD, TA-CS-SD(1:10), PM and (B) TA-CS-SD(1:5), TA-CS-SD(1:10), TA-CS-SD(1:20), TA-CS-SD(1:40).
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3. Figure 3. DSC thermograms of TA, CS-SD, TA-CS-SD(1:10), and PM.
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4. Figure 4. FTIR spectrums of TA, CS-SD, TA-CS-SD(1:10), and PM. 5. Figure 5. SEM micrographs of (A) TA, (B) CS-SD, (C) PM, (D) TA-CS-SD(1:5), (E) TA-CS-SD(1:10), and (F) TA-CS-SD(1:20).
6. Figure 6. Degradation (A) and Swelling (B) profiles of CS-SD.
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7. Figure 7. Percent A549 cell viability (% control) treated 24 h with the different formulations: blank medium, DMSO, CS-SD dispersed in medium, TA dispersed in medium, TA dissolved in DMSO, and TA-CS-SD (1:10) dispersed in medium
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respectively (n = 5). *P < 0.05, **P < 0.01 and ***P < 0.001 compared with TA
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dispersed in medium.
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1. A novel tanshinone IIA solid dispersion is fabricated using chitosan as carrier for
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the first time. 2. The dissolution rate of tanshinone IIA is improved by solid dispersion approach. 3. The release mechanism of solid dispersion exhibits pH-responsive performance.
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4. The crystallinity of tanshinone IIA is decreased by solid dispersion approach, and
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the reduction of crystallinity is correlated with the content of chitosan matrix. 5. In vitro cytotoxicity test is performed to verify the bioactivity improvement of
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tanshinone IIA solid dispersion.
S1773-2247(18)31211-5
DOI:
https://doi.org/10.1016/j.jddst.2018.11.024
Reference:
JDDST 846
To appear in:
Journal of Drug Delivery Science and Technology
Received Date: 15 October 2018 Revised Date:
18 November 2018
Accepted Date: 25 November 2018
Please cite this article as: C. Luo, W. Wu, X. Lin, Y. Li, K. Yang, A novel tanshinone IIA/chitosan solid dispersion: Preparation, characterization and cytotoxicity evaluation, Journal of Drug Delivery Science and Technology (2018), doi: https://doi.org/10.1016/j.jddst.2018.11.024. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT A novel tanshinone IIA/chitosan solid dispersion: preparation, characterization and cytotoxicity evaluation Chao Luo a, Weibin Wu b, Xinyu Lin a, Yaoqi Li a, Kai Yang c, * a
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Department of Medicine and Health, Shaoxing University Yuanpei College, Shaoxing, China, 312000; b Department of Basic Medicine, Zhaoqing Medical College, Zhaoqing, China, 526020; c State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, Guangdong Key Laboratory of Vascular Disease, Guangzhou Institute of Respiratory Health, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou, China, 510182.
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Running title: A novel tanshinone IIA/chitosan solid dispersion *
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Address correspondence to: Kai Yang, Ph.D. Associate Professor of Medicine State Key Laboratory of Respiratory Disease Guangzhou Institute of Respiratory Health The First Affiliated Hospital of Guangzhou Medical University 195 Dong Feng Xi Road, Guangzhou, Guangdong, China, 510182 Email: [email protected]
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Author Contributions: C.L. initiated and designed the project, performed the experiments, analyzed data and wrote the manuscript; W.W., X.L., and Y.L. performed the experiments; K.Y. contributed to the design of the project, provided consultation and edited the manuscript. All authors accepted the submission of the manuscript.
ACCEPTED MANUSCRIPT ABSTRACT The objective of this study was to develop and evaluate a novel tanshinone IIA (TA)/chitosan (CS) solid dispersion (TA-CS-SD) to improve the bioavailability. Solid
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dispersion of tanshinone IIA with chitosan was prepared by a pH inversion method via the pH-responsive dissolution property of chitosan matrix. The in vitro dissolution study confirmed dramatically elevated dissolution rate of TA by solid dispersion
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approach, and the release mechanism exhibited pH-responsive performance. The solid
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dispersion was characterized by x-ray diffractometry (XRD), differential scanning calorimetry (DSC), fourier-transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM). Our results showed that TA existed in the form of microcrystal or superfine crystal in solid dispersion, and the reduction of crystallinity
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was correlated with the content of CS matrix. In vitro cytotoxicity study was performed in human non-small cell lung cancer A549 cell line by CCK-8 assay. Significant improvement in antitumor activity of TA in TA-CS-SD was achieved
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compared to the crystalline drug. These results indicated that, compared with free TA,
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TA-CS-SD demonstrated higher dissolution rate and bioactivity. Keywords: Tanshinone IIA, Chitosan, Solid dispersion, pH-responsive, cytotoxicity
ACCEPTED MANUSCRIPT 1. INTRODUCTION Tanshinone IIA (TA), a lipid-soluble bioactive ingredient extracted and purified from the root of Salvia miltiorrhiza Bunge (Danshen) [1], which has been widely used the
treatment
of
cardiovascular
disorders,
cerebrovascular
diseases,
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for
neurodegenerative diseases, pulmonary hypertension and cancer [2-8]. However, the clinical application of TA is hindered by its poor water-solubility (2.8 ng·mL-1) [9],
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short half-life (1-2 h) [10], first pass metabolism [11], and low oral bioavailability.
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Currently, numerous new drug delivery systems have been employed to overcome these issues, such as the nanoparticles [12], polymeric micelles [13], micro-emulsions [14], solid dispersion (SD) [15], intravenous lipid emulsion [16], etc. SD is a highly effective strategy for improving the solubility, dissolution, and
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bioavailability of poorly water-soluble drugs [17]. The mechanisms of increasing drug solubility by SD technology is due to reduced particle size [18], improved wettability [19], increased porosity [20], and alteration in the polymorphic state of drugs from
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crystalline to an amorphous form [21]. Synthetic polymer materials, such as
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polyethylene glycols (PEG) [22], polyvinylpyrrolidone (PVP) [23], hydroxypropyl methylcellulose (HPMC) [24], poloxamers [25], have been widely used in solid dispersion for improving dissolution characteristics and bioavailability of poorly aqueous-soluble drugs. Nowadays, the application of bio-macromolecules, such as alginate [26], chitosan (CS) [27], and cellulose [28], as the carriers for SD has been widely tested and proved to be ideal vehicles. CS, a natural biopolymer derived from chitin and is the second rich nature
ACCEPTED MANUSCRIPT polysaccharide after cellulose, has been widely used as carriers of drug delivery system due to its favorable chemical and biological properties, such as biocompatibility, biodegradability, pH-responsive, adsorptivity, and its hydrophilic
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and mucoadhesive nature [29-31]. Moreover, based on its excellent characteristics, CS has attracted more and more attention as a carrier for hydrophobic drugs to increase the solubility, dissolution, and bioavailability. The CS-based drug delivery carriers,
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such as microspheres [32], micelles [33] and nanoparticles [34], have been reported as
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supporters for loading hydrophobic drugs, Jain et al. [35] fabricated a docetaxel loaded CS nanoparticle, which represents a 25% increase in cytotoxicity compared with the free drug. And recently, more convenient methods such as SD, direct adsorption of powder are also fabricated for loading hydrophobic drugs on CS [36].
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For example, Crucitti et al. [27] reported an abietic acid/chitosan solid dispersion, which can improve the water solubility of abietic acid and promote its bioactivity. To date, there is no report about using CS as carrier for fabricating the solid
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dispersion of TA. Based on the successful development of CS-SD system and given
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the long history of successful usage of TA in clinical treatment of substantial diseases, here in this study, for the first time, we develop a novel tanshinone IIA/chitosan solid dispersion (TA-CS-SD) by using chitosan as carrier to improve the dissolution rate of TA, which provides sustained release performance and pH-sensibility. It is well known that chitosan is insoluble at neutral or slightly alkaline pH and highly soluble at acidic environment [37], and can be regenerated with pH inversion [38]. So, in this study, a rapid one-pot approach for TA-CS-SD is developed by inverting pH of the
ACCEPTED MANUSCRIPT chitosan acetic acid aqueous dispersed with TA. TA is dissolved in ethyl alcohol, and then mixed with chitosan solution, the viscous polymer solution can effectively disperse and adsorb the TA molecules, after that, with the regeneration of CS, the TA
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is coated into the CS matrix, and homogeneous dispersion. The dissolution rate improvement and sustained release performance of the TA-CS-SD are evaluated. The scanning electron microscopy (SEM) is used to examine the morphological structure
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of the TA-CS-SD. The physical and chemical compatibility between the TA and CS in
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SD are characterized by fourier-transform infrared spectroscopy (FTIR). X-ray diffraction (XRD) and differential scanning calorimeter (DSC) are employed to demonstrate that the crystallization degree of TA is decreased after encapsulated into chitosan matrix. Subsequently, in vitro degradation research and swelling experiment
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are performed to assess the pH-sensitive of the TA-CS-SD. The human non-small cell lung cancer A549 cell line is used for the cytotoxicity test to verify the bioactivity
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improvement of solid dispersion.
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2. MATERIALS AND METHODS 2.1 Materials
Pharmaceutical grade, 95% deacetylated chitosan with a viscosity average
molecular weight of 1.2 × 105 was supplied by Aoxing Biotechnology Co., Ltd. (Zhejiang, China). Tanshinone IIA (98%) was purchased from Hao Xuan biological Co., Ltd. (Xi’an, China). Methanol (HPLC grade) was purchased from Aladdin Industrial Corporation (Shanghai, China). Deionized water was used throughout,
ACCEPTED MANUSCRIPT produced by Milli-Q Reference Water Purification System (Merck Millipore, USA). All other chemicals were purchased from commercial sources and used as received. The human non-small cell lung cancer A549 cell line was purchased from the
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Cell Bank of the Chinese Academy of Sciences (Shanghai, China). RPMI1640 medium and fetal bovine serum (FBS) were supplied by Gibco (Carlsbad, USA). Glutamine was from Sigma-Aldrich Trade Co. (USA). The Cell Counting Kit-8
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(CCK-8) was purchased from Beyotime Biotechnology Co (Shanghai, China).
2.2 Preparation of TA-CS-SD and physical mixture (PM)
TA-CS-SD was prepared using the pH inversion method with TA and CS at different mass ratio (1:5, 1:10, 1:20 and 1:40). Briefly, two solutions were prepared: in
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solution A, certain amounts of CS (0.25, 0.5, 1.0, 1.5, 2.0 g, respectively) were dissolved in 100 ml 1 wt% acetic acid solution under magnetic stirring, and in solution B, 50 mg of TA was dissolved in anhydrous alcohol with a final
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concentration of 1 mg/mL. Then, Solution B was added slowly into solution A with
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stirring at 1000 rpm for 30min. subsequently, the resultant mixture solution was neutralized with 1 wt% NaOH solution, washed to neutral with deionized water, and then freeze-dried. The yield of the obtained TA-CS-SD was calculated by the following formula, and the final products were stored in a desiccator for further experiments. The regenerative CS was prepared following the above methods except for the addition of TA, named CS-SD. The physical mixture (PM) was prepared by mixing TA and CS-SD at the weight ratio of 1:10.
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% =
× 100%
2.3 Analytical methodology
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The concentration of TA in the testing medium were quantified by a Waters e2695-2489 liquid chromatographic system (HPLC) under the following conditions: SunFire C18 (4.6mm ×250 mm, 5 µm) column, methanol and water (90:10 v/v) as
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mobile phase at a flow rate of 1.0 ml/min, UV detection at 270 nm. The linearity of
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the method was investigated in the concentration range of 1.0-20.0 µg/ml (r=0.9996). The RSD of intraday and interday precision for TA were below 2%.
2.4 Encapsulation efficiency and dissolution test in vitro
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A 5.0 mg of TA-CS-SD was dissolved in 10.0 ml of 0.1 M hydrochloric acid solution (pH 1.2) and centrifuged at 12000 rpm for 10 min, and 1.0 ml of the supernatant was diluted to 10.0 ml with anhydrous alcohol. The TA in the solution
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was determined by HPLC at wavelength of 270 nm. The drug loading content and
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encapsulation efficiency were calculated via the following equations: 2 $%& '( )& *')+ )+ % =
3 e)*(34% (+ ') 55 * )*6 % =
,
,-./-/0
,-./-/0 ,
× 100%
,-./-/0
,
× 100%
The in vitro dissolution study was performed using USP type II (paddle type apparatus) at 100 rpm, 37 ± 0.5 °C using 900 ml of simulated intestinal fluid (pH 7.4 phosphate buffer solution contained 0.5% sodium dodecyl sulfate) or simulated
ACCEPTED MANUSCRIPT gastric fluid (0.1 mol/L HCl, pH 1.2) as the dissolution medium. Briefly, TA-CS-SD or PM equivalent to 5.0 mg TA were sealed into dialysis bag and immersed in dissolution medium. At pre-determined time points, 5 mL of the sample was
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withdrawn and the equal volume of dissolution medium was instantly replaced to maintain a constant dissolution volume. The samples were filtered through 0.45 µm millipore filter and analyzed by HPLC as described above. Experiments were carried
2.5 Characterization of TA-CS-SD
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out in triplicate.
2.5.1 Powder X-ray diffraction (XRD)
The XRD was performed on a X'pert PRO X-ray diffraction system (PANalytical,
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Netherlands) with Cu Kα radiation, at a voltage of 40 kV and a current of 40 mA. The scanning rate was 2°/min over a 2θ range of 5–60°.
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2.5.2 Differential scanning calorimetry (DSC)
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The thermal behavior of TA-CS-SD and PM were performed with a Mettler Toledo TGA/DSC 2 STARe system. Samples were crimped in aluminium crucible and heated at a rate of 20 °C/min from 25 °C to 300 °C. Nitrogen was used as the purge gas at a flow rate of 20 mL/min.
2.5.3 Fourier-transform infrared spectroscopy (FTIR) The FTIR analysis was performed with a Nicolet 740 spectrometer (Thermo
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2.5.4 Scanning Electron Microscopy (SEM)
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range of 4000-400 cm-1.
The morphology of TA-CS-SD, PM, TA and CS-SD was observed with a
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JEM-6360 SEM. The samples were mounted on metal stubs using adhesive tape and
SEM.
2.6 Degradation studies
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made electrically conductive by sputter-coating with gold and then observed with
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The in vitro degradation of CS-SD was carried out in 0.1 M HCl solution (pH 1.2). CS-SD sample was dispersed in 10.0 ml of buffer, incubated in a 37 ℃ shaker at 150 r/min. At regular intervals, the samples were placed in the ultra-filtration
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centrifuge tubes (MWCO 50K), centrifuged at 12000 rpm for 15 min, and dried in an
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oven at 60 ℃ to a constant weight. The remaining CS-SD was weighed, and the results were represented as undegraded mass percent versus time. The experiment was performed in triplicate.
2.7 Swelling studies The swelling ratio of CS-SD was carried out in PBS (pH 7.4) at 37 ℃. At regular intervals, the swollen CS-SD was weighed after the excess liquid were carefully
ACCEPTED MANUSCRIPT wiping off with a tissue paper. The swelling ratio (Sw) was calculated by the following equation, where Wd and Ws are the weights of the dried and swollen CS-SD, respectively. Three replicate tests were performed in the swelling studies. % =
9 −9 × 100% 9
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4 8
2.8 Cell viability
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A549 cells were cultured in RPMI1640 medium, supplemented with 10% (V/V)
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FBS and 2 mM glutamine in a humidified incubator at 37°C, 5% CO2 atmosphere. Fresh medium was replaced every 48 h, cells in logarithmic phase will be used in the experiment. TA and TA-CS-SD (1:10) were dispersed in medium at various concentrations (2.5, 5, 10 µM), and TA dissolved in DMSO and diluted by medium at
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the same concentrations were used as comparisons.
Cell proliferation level was determined by CCK-8 assay. Briefly, A549 cells were seeded in 96-well plates at a density of 1×105 cells/0.1ml and cultured for 24 h
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(37 °C, 5% CO2). Then, the medium was removed and cells were treated with
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formulation equivalent to TA at various concentrations (2.5, 5, 10 µM). Medium, DMSO (diluted by medium), and CS-SD (dispersed in medium, 35 µg/ml) were used as control groups, the amount of CS-SD used in cytotoxicity test is equivalent to the mass of CS matrix in TA-CS-SD of high concentration group. The plates were then incubated (37 °C, 5% CO2) for 24 h, and the medium was replaced by 90 µl of basal medium and 10 µl of CCK-8, and continue to incubate for another 4 h. Afterwards, the cell viability was detected by scanning with ELISA plate reader (Thermo
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individual experiments were performed and analyzed in each group.
2.9 Statistical analysis
The results were represented as mean ± standard deviation (mean ± SD).
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Statistical analysis was performed with SPSS 17.0 software. One way analysis of
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variance (ANOVA) and LSD test were used for data analysis. P < 0.05 was considered as statistically significant.
3. RESULTS AND DISCUSSION
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3.1 Drug loading and encapsulation efficiency
Melting method and solvent evaporation method are the two major methods for preparation of SD [39]. The main advantages of these two methods are to offer a
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simple preparation process and can obtain the well yield and encapsulation efficiency.
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However, the melting method requires a high thermal stability of matrix, and the solvent evaporation method needs the well lipophilicity of matrix. Therefore, these methods are not suitable for the preparation of CS-SD. Actually, CS can be regenerated from its acidic solution with pH inversion, and this property can be employed to prepare the CS-SD. In order to evaluate the efficiency of this method for preparing SD, the yield and encapsulation efficiency were firstly studied. The yield, encapsulation efficiency and drug loading of TA-CS-SD with different
ACCEPTED MANUSCRIPT TA and CS mass ratio are shown in Table 1. As seen, the method used in preparation of TA-CS-SD is characterized by high yield and encapsulation efficiency. As the mass ratio of TA and CS increases from 1:5 to 1:10, the encapsulation efficiency of the
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TA-CS-SD increases from 84.71 to 94.55%, and stays at a plateau of 93-94%, likely due to the adsorption capacity of CS. The CS macromolecules in solution can adsorb and disperse the TA, and prevent its crystallization. As the solid dispersion is prepared
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with a higher amount of CS, it has a stronger adsorption and dispersion capacity, and
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represents a higher encapsulation efficiency. However, the drug loading of the TA-CS-SD is decreased from 15.69% to 2.36%, with the mass ratio of TA and CS increase from 1:5 to 1:40, respectively, which is caused by the increase in the
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proportion of the matrix.
3.2 Dissolution behavior of TA-CS-SD
To evaluate the pH-dependent release behavior of the TA-CS-SD, in vitro release
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assay was performed in the simulated intestinal fluid (pH 7.4) and simulated gastric
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fluid (pH 1.2). As is shown in Fig. 1, the release of TA from the solid dispersion represented a quite different release pattern of TA-CS-SD at pH 7.4 and 1.2. In compare with pH 7.4, the drug showed a faster release rate and higher dissolution proportion at pH 1.2, which can be explained by the pH-responsive of CS matrix. CS is highly soluble at acidic environment, but insoluble at neutral or slightly alkaline pH. This hypothesis can be supported by the results of swelling and degradation tests. In the dissolution medium at pH 1.2 (Fig. 1A), the release rate of TA from the solid
ACCEPTED MANUSCRIPT dispersion is decreased consistently with increasing ratio of TA: CS from 1:5 to 1:40 due to the dissolution time of chitosan matrix. PM has a poor dissolution rate in dissolution medium due to the high crystallinity of TA, the cumulative release amount
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in 210 min is below 1%, but the dissolution rate of TA in SD increases significantly, the cumulative release amount within 210 min is up to nearly 100% (except for the group of TA: CS with 1:5). It can be explained that the crystallinity of TA is declined
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following the SD process, and the increased amount of CS matrix may prevent the
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crystallization of TA. These results are further supported by data from XRD and SEM. Whereas at a pH of 7.4 (Fig. 1B), the TA-CS-SD has a well sustained-release performance, with increasing ratio of TA: CS from 1:5 to 1:40, the cumulative release amount at 24 h is decreased from 48% to 32%. The mechanism of TA release from SD
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at pH 7.4 may be predominantly diffusion-controlled, and the swelling property of the CS matrix is the main factor to control the dissolution rate [40]. Collectively, the encapsulation efficiency, drug loading, and dissolution rate are correlated with the
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ratios of drug: polymer in the formulation. Because of the higher encapsulation
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efficiency, drug loading, and dissolution rate, the group of TA-CS-SD (1:10) was selected as a sample used in characterization and in vitro cytotoxicity.
3.3 Characterization
3.3.1 Powder X-ray diffraction (XRD) The XRD patterns of TA, CS, TA-CS-SD, and PM are shown in Fig. 2. The diffraction peaks of CS at around 10° and 20° are characteristic of the hydrated
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retains the distinctive peaks attributed to the crystalline structure of TA. Compared to PM, the characteristic reflection peak of TA at 2θ 7.20° still remains in the TA-CS-SD, but the relative intensity is lower. This may suggest that TA is still in the form of
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microcrystal in TA-CS-SD, but the crystal growth of TA may be prohibited by CS to
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some extent. Therefore, the crystallinity of TA in TA-CS-SD is relatively lower [15]. Furthermore, with increasing ratio of TA: CS from 1:5 to 1:40, the intensity of the characteristic peak of TA decreased gradually, indicating the reduction of crystallinity is correlated with the content of CS matrix (Fig. 2B). These results can explain the
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high solubility of TA in SD.
3.3.2 Differential scanning calorimetry (DSC)
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As seen in Fig. 3, the CS thermogram shows a wide endothermic band centered
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at approximately 75 °C due to the presence of water. The DSC curve of TA shows a sharp endothermic peak at 219.20 °C corresponding to its melting point, indicating its nature of crystalline state. Because of the high content of CS, it is difficult to discern the endothermic peak of TA in TA-CS-SD and PM [27]. But the endothermic event of TA in TA-CS-SD is altered from 219.20°C to 197.77°C. It may be due to the formation of eutectic mixtures in TA-CS-SD leading to the depression of melting point, and this result also suggests that a reduction of the crystallinity of TA [15],
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3.3.3 Fourier-transform infrared spectroscopy (FTIR) FTIR spectroscopy was applied to investigate the interactions between TA and CS in solid dispersion. As shown in Fig. 4, CS shows characteristic peaks at 3148
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cm-1 (O-H and N-H stretching vibration), 1650 cm−1 (C=O stretching vibration (amide
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I)), 1578 cm−1 (N-H in-plane bending vibration (amide II)), and 1095 cm−1 (C-O stretching vibration) [42]. For TA, the main characteristic peak is found at 1670 cm-1 (C=O stretching vibration), and the peaks at 2925 and 2866cm-1 are assigned to the C-H stretching vibration of -CH2, and -CH3 [43]. The FTIR spectra of the PM
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comprise a summation of TA and CS. The FTIR spectra of the TA-CS-SD are similar to the spectra of mixture, but many weaker peaks of TA are covered by CS, indicating that TA is uniformly dispersed in the CS matrix, and the quantity of CS is more than
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that of TA, so the signals of CS overshadow many weaker peaks from smaller
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quantities of TA. The results suggest that there are no chemical interactions between TA and carriers.
3.3.4 Morphological analysis The morphology of pure TA, CS, PM, and TA-CS-SD at drug/polymer weight ratios of 1:5, 1:10, and 1:20 were studied via SEM to investigate the potential morphological differences (Fig. 5). The crystal morphology of pure TA is an acicular
ACCEPTED MANUSCRIPT crystal, CS has an irregular shape. Morphology of the PM displayed the crystals of TA and polymer in the mixture. These results confirm the presence of crystalline TA in PM. Conversely, the crystals of TA are invisible in TA-CS-SD at the weight ratios of
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1:10 and 1:20, the TA-CS-SD appears in the form of irregular particles. But, for TA-CS-SD (1:5), the small crystalline TA was observed at the surface of the solid dispersion. These results in combine demonstrated that TA may disperse in solid
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dispersion in microcrystal form, and the crystal growth of TA can be prohibited by CS,
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furthermore, the prohibition effect may be related to the mass ratio of TA and CS. This finding is in accordance with the results of XRD.
3.4 Degradation and Swelling studies
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To elucidate the different release mechanism of SD in different pH, the degradation study and swelling study of CS-SD were conducted in simulated gastric fluid (pH 1.2) and simulated intestinal fluid (pH 7.4). As shown in Fig. 6A, CS-SD is
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almost degraded completely within 90 min, likely due to the well solubility of CS at
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acidic environment. Conversely, CS is insoluble at slightly alkaline pH, but it has well swelling property due to the hydrophilic groups on the CS, the mass of CS-SD increase by about 3-fold within 10 h (Fig. 6B), indicating that TA-CS-SD has different release mechanism in different pH environment, the drug release is related to the degradation of the CS matrix in simulated gastric fluid (pH 1.2), but in simulated intestinal fluid (pH 7.4), the swelling process might be a key factor to control the drug release. These results are in agreement with the previous study [40].
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3.5 Cell viability To evaluate the antitumor activity improvement of TA after it was prepared into
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solid dispersion, the in vitro cytotoxicity was performed using the human non-small cell lung cancer A549 cell line as a cellular model. As is shown in Fig. 7, there is no obvious cell cytotoxicity of the CS-SD group, indicating excellent biocompatibility
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and negligible cytotoxicity of CS matrix. Because of the poor water solubility, the
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group of TA dispersed in medium has little activity on A549. In contrast, the group of TA dissolved in DMSO significantly inhibits the growth of A549 in a dose-dependent manner with a percent decrease of cell viability of 15%, 25%, 37 %, respectively. The cytotoxicity of TA-CS-SD (1:10) is similar to the group of TA dissolved in DMSO at
4. CONCLUSION
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the same concentrations, likely due to the increased solubility of TA in SD.
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In this study, a novel solid dispersion of TA by using CS as a carrier was
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successfully developed. Our data showed that TA-CS-SD prepared by pH inversion method can largely increase the dissolution rate of TA, and the release mechanism of SD exhibited a pH-responsive way. The TA-CS-SD was characterized by XRD, DSC, FTIR, and SEM, with all confirming that TA is present in the form of microcrystal or superfine crystal in SD, and the reduction of crystallinity is correlated with the content of CS matrix. No interactions of drug substances and the carriers are found in SD. Moreover, the antitumor activity of TA, reflected by in vitro cytotoxicity test, is
ACCEPTED MANUSCRIPT significantly enhanced in TA-CS-SD. In conclusion, the SD using CS as a carrier can markedly improve the dissolution rate and bioactivity of TA, and the preparation process of TA-CS-SD, for the first, provides a novel approach to overcoming the low
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dissolution rate and activity of poorly water-soluble drugs, such as TA, and has the potential to largely increase the clinical usage of this group of well-proved clinical
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medication.
ACCEPTED MANUSCRIPT Table 1. The yield, encapsulation efficiency and drug loading of the prepared TA-CS-SD. Encapsulation efficiency
Drug loading
(%)
(%)
(%)
TA-CS-SD(1:5)
90.11±5.05
84.71±2.18
TA-CS-SD(1:10)
97.03±2.96
94.55±3.88
TA-CS-SD(1:20)
97.56±0.88
92.97±4.44
TA-CS-SD(1:40)
97.27±0.40
94.05±3.55
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Yield
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Sample
15.69±0.59 8.86±0.13 4.54±0.21 2.36±0.08
ACCEPTED MANUSCRIPT Declaration of interest The authors disclose no potential conflicts of interest.
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Acknowledgements The authors acknowledge the financial support from the Natural Science Foundation of Zhejiang province (LQ16H310002) and the National Natural Science
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Foundation of China (81800057).
ACCEPTED MANUSCRIPT Reference [1] S. Gao, Z.P. Liu, H. Li, P.J. Little, P.Q. Liu, S.W. Xu, Cardiovascular actions and therapeutic potential of tanshinone IIA, Atherosclerosis. 220 (2012) 3-10.
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[2] J.Y. Han, J.Y. Fan, Y. Horie, S. Miura, D.H. Cui, H. Ishii, T. Hibi, H. Tsuneki, I. Kimura, Ameliorating effects of compounds derived from Salvia miltiorrhiza root
reperfusion, Pharmacol Ther. 117 (2008) 280-295.
SC
extract on microcirculatory disturbance and target organ injury by ischemia and
M AN U
[3] T. Song, Y. Yao, T. Wang, H. Huang, H. Xia, Tanshinone IIA ameliorates apoptosis of myocardiocytes by up-regulation of miR-133 and suppression of Caspase-9, Eur J Pharmacol. 815(2017) 343-350.
[4] X.H. Wang, S.L. Morris-Natschke, K.H. Lee, New developments in the chemistry
133-148.
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and biology of the bioactive constituents of Tanshen, Med Res Rev. 27 (2007)
[5] J.G. Wang, S.C. Bondy, L. Zhou, F.Z. Yang, Z.G. Ding, Y. Hu, Y. Tian, P.Y. Wen,
EP
H. Luo, F. Wang, W.W. Li, J. Zhou, Protective effect of tanshinone IIA against infarct
AC C
size and increased HMGB1, NFkappaB, GFAP and apoptosis consequent to transient middle cerebral artery occlusion, Neurochem Res. 39(2014) 295-304. [6] X. Liu, M. Ye, C.Y. An, L.Q. Pan, L.T. Ji, The effect of cationic albumin-conjugated PEGylated tanshinone IIA nanoparticles on neuronal signal pathways and neuroprotection in cerebral ischemia, Biomaterials. 34 (2013) 6893-6905. [7] S.C. Chiu, S.Y. Huang, S.P. Chen, C.C. Su, T.L. Chiu, C.Y. Pang, Tanshinone IIA
ACCEPTED MANUSCRIPT inhibits human prostate cancer cells growth by induction of endoplasmic reticulum stress in vitro and in vivo, Prostate Cancer Prostatic Dis. 16(2013) 315-322. [8] C. Lin, L. Wang, H. Wang, L. Yang, H. Guo, X. Wang, Tanshinone IIA inhibits
RI PT
breast cancer stem cells growth in vitro and in vivo through attenuation of IL-6/STAT3/NF-κB signaling pathways, J Cell Biochem. 114(2013) 2061-2070.
[9] H.X. Yan, J. Li, Z.H. Li, W.L. Zhang, J.P. Liu, Tanshinone IIA – loaded pellets
SC
developed for angina chronotherapy: Deconvolution-based formulation design and
M AN U
optimization, pharmacokinetic and pharmacodynamic evaluation, Eur J Pharm Sci. 76(2015) 156-164.
[10] W.L. Zhang, H.L. He, J.P. Liu, J. Wang, S.Y. Zhang, S.S. Zhang, Z.M. Wu, Pharmacokinetics and atherosclerotic lesions targeting effects of tanshinone IIA
306-319.
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discoidal and spherical biomimetic high density lipoproteins, Biomaterials. 34(2013)
[11] Y. Zhang, P.X. Jiang, M. Ye, S. H. Kim, C. Jiang, J.X. Lü, Tanshinones: sources,
EP
pharmacokinetics and anti-cancer activities, Int J Mol Sci. 13(2012) 13621-13666.
AC C
[12] Q. Li, Y. Wang, N. Feng, Z. Fan, J. Sun, Y. Nan, Novel polymeric nanoparticles containing tanshinone IIA for the treatment of hepatoma, J Drug Target. 16(2008) 725-732.
[13] J.M. Zhang, Y.B. Li, X.F. Fang, D.M. Zhou, Y.T. Wang, M.W. Chen, TPGS-g-PLGA/Pluronic F68 mixed micelles for tanshinone IIA delivery in cancer therapy, Int J Pharm. 476 (2014) 185-198. [14] H. Ma, Q. Fan, J. Yu, J. Xin, C. Zhang, Novel microemulsion of tanshinone IIA,
ACCEPTED MANUSCRIPT isolated from Salvia miltiorrhiza Bunge, exerts anticancer activity through inducing apoptosis in hepatoma cells, Am J Chin Med. 41 (2013) 197-210. [15] X.F. Zhai, C.G. Li, G.B. Lenon, C.L. Xue, W.Z. Li, Preparation and
RI PT
characterisation of solid dispersions of tanshinone IIA, cryptotanshinone and total tanshinones, Asian J Pharm Sci. 12 (2017) 85-97.
[16] T. Chu, Q. Zhang, H. Li, W.C. Ma, N. Zhang, H. Jin, S.J. Mao, Development of
M AN U
activity in vitro, Int J Pharm. 424 (2012) 76-88.
SC
intravenous lipid emulsion of tanshinone IIA and evaluation of its anti-hepatoma
[17] S. Lee, K. Nam, M.S. Kim, S.W. Jun, J.S. Park, J.S. Woo, S.J. Hwang, Preparation and characterization of solid dispersions of itraconazole by using aerosol solvent extraction system for improvement in drug solubility and bioavailability, Arch
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Pharm Res. 28 (2005) 866-74.
[18] S. Janssens, G. Van den Mooter, Review: physical chemistry of solid dispersions, J Pharm Pharmacol. 61 (2009) 1571-1586.
EP
[19] N. Zerrouk, C. Chemtob, P. Arnaud, S. Toscani, J. Dugue, In vitro and in vivo
AC C
evaluation of carbamazepine-PEG 6000 solid dispersions, Int. J. Pharm. 225 (2001) 49-62.
[20] J. Li, P. Liu, J.P. Liu, W.L. Zhang, J.K. Yang, Y.Q. Fan, Novel Tanshinone II A ternary solid dispersion pellets prepared by a single-step technique: In vitro and in vivo evaluation, Eur J Pharm Biopharm. 80 (2012) 426-432. [22] D.P. Otto, A. Otto, M. M. Villiers, Experimental and mesoscale computational dynamics studies of the relationship between solubility and release of quercetin from
ACCEPTED MANUSCRIPT PEG solid dispersions, Int. J. Pharm. 456 (2013) 282-292. [23] A. Jahangiri, M. Barzegar-Jalali, A. Garjani, Y. Javadzadeh, H. Hamishehkar, A. Afroozian,
K.
Adibkia,
Pharmacological
and
histological
examination
of
RI PT
atorvastatin-PVP K30 solid dispersions, Powder Technol. 286 (2015) 538-545. [24] M.K. Riekes, G. Kuminek, G.S. Rauber, C. Eduardo, M. Campos, A.J. Bortoluzzi, H.K. Stulzer, HPMC as a potential enhancer of nimodipine biopharmaceutical
SC
properties via ball-milled solid dispersions, Carbohyd Polym. 99 (2014) 474-482.
M AN U
[25] J.O. Eloy, J.M. Marchetti, Solid dispersions containing ursolic acid in Poloxamer 407 and PEG 6000: A comparative study of fusion and solvent methods, Powder Technol. 253 (2014) 98-106.
[26] J. Guan, Q. Liu, X. Zhang, Y. Zhang, R. Chokshi, H. Wu, S. Mao, Alginate as a
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potential diphase solid dispersion carrier with enhanced drug dissolution and improved storage stability, Eur J Pharm Sci. 114 (2018) 346-355. [27] V.C. Crucitti, L.M. Migneco, A. Piozzi, V. Taresco, M. Garnett, R.H. Argent, I.
EP
Francolini, Intermolecular interaction and solid state characterization of abietic
AC C
acid/chitosan solid dispersions possessing antimicrobial and antioxidant properties, Eur J Pharm Biopharm. 125 (2018) 114-123. [28] B. Li, S. Konecke, L.A. Wegiel, L.S. Taylor, K.J. Edgar, Both solubility and chemical stability of curcumin are enhanced by solid dispersion in cellulose derivative matrices, Carbohyd Polym. 98 (2013) 1108-1116. [29] M.A. Pujana, L. Pérez-Álvarez, L.C. Iturbe, I. Katime, Biodegradable chitosan nanogels crosslinked with genipin, Carbohyd Polym. 94 (2013) 836-842.
ACCEPTED MANUSCRIPT [30] E. Yilmaz, Z. Yalinca, K. Yahyaa, U. Sirotina, pH responsive graft copolymers of chitosan, Int J Biol Macromol. 90 (2016) 68-74. [31] K. Kim, K. Kim, J.H. Ryu, H. Lee, Chitosan-catechol: A polymer with
RI PT
long-lasting mucoadhesive properties, Biomaterials. 52 (2015) 161-170. [32] A.J. Ribeiro, R.J. Neufeld, P. Arnaud, J.C. Chaumeil, Microencapsulation of lipophilic drugs in chitosan-coated alginate microspheres, Int. J. Pharm. 187 (1999)
SC
115-123.
M AN U
[33] J.P. Nam, S.C. Park, T.H. Kim, J.Y. Jang, C. Choi, M.K. Jang, J.W. Nah, Encapsulation of paclitaxel into lauric acid-O-carboxymethyl chitosan-transferrin micelles for hydrophobic drug delivery and site-specific targeted delivery, Int. J. Pharm. 457 (2013) 124-135.
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[34] L.H.Chuah, C.J. Roberts, N. Billa, S. Abdullah, R. Rosli, Cellular uptake and anticancer effects of mucoadhesive curcumin-containing chitosan nanoparticles, Colloids Surf B. 116 (2014) 228-236.
EP
[35] A. Jain, K. Thakur, G. Sharma, P. Kush, U.K. Jain, Fabrication, characterization
AC C
and cytotoxicity studies of ionically cross-linked docetaxel loaded chitosan nanoparticles, Carbohydr Polym. 137 (2016) 65-74. [36] D. Liudvinaviciute, K. Almonaityte, R. Rutkaite, J. Bendoraitiene, R. Klimaviciute, Adsorption of rosmarinic acid from aqueous solution on chitosan powder, Int J Biol Macromol. 118 (2018) 1013-1020. [37] S. Abbad, Z. Zhang, A.Y. Waddad, W.L. Munyendo, H. Lv, J. Zhou, Chitosan-modified cationic amino acid nanoparticles as a novel oral delivery system
ACCEPTED MANUSCRIPT for insulin, J Biomed Nanotechnol. 11 (2015) 486-499. [38] S. Bi, Z. Bao, X. Bai, S. Hu, X. Cheng, X. Chen, Tough chitosan hydrogel based on purified regeneration and alkaline solvent as biomaterials for tissue engineering
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applications, Int J Biol Macromol. 104 (2017) 224-231. [39] C.L. Vo, C. Park, B.J. Lee, Current trends and future perspectives of solid dispersions containing poorly water-soluble drugs, Eur J Pharm Biopharm. 85 (2013)
SC
799-813.
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[40] S. Hua, H. Yang, W. Wang, A. Wang, Controlled release of ofloxacin from chitosan–montmorillonite hydrogel, Appl Clay Sci. 50 (2010) 112-117. [41] A.F. Martins, D.M. de Oliveira, A.G.B. Pereira, A.F. Rubira, E.C. Muniz, Chitosan/TPP microparticles obtained by microemulsion method applied in controlled
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release of heparin, Int J Biol Macromol. 51 (2012) 1127-1133. [42] B.R. dos Santos, F.B. Bacalhau, T.S. Pereira, C.F. Souza, R. Faez, Chitosan-Montmorillonite microspheres: A sustainable fertilizer delivery system,
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Carbohydr Polym. 127 (2015) 340-346.
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[43] Z. Meng, L. Meng, K. Wang, J. Li, X. Cao, J. Wu, Y. Hu, Enhanced hepatic targeting, biodistribution and antifibrotic efficacy of tanshinone IIA loaded globin nanoparticles, Eur J Pharm Sci. 73 (2015) 35-43.
ACCEPTED MANUSCRIPT Captions 1. Figure 1. Dissolution behavior of TA-CS-SD at different drug: carrier ratios in different medium; (A) simulated gastric fluid (pH1.2) and (B) simulated intestinal
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fluid (pH7.4). 2. Figure 2. XRD patterns of (A) TA, CS-SD, TA-CS-SD(1:10), PM and (B) TA-CS-SD(1:5), TA-CS-SD(1:10), TA-CS-SD(1:20), TA-CS-SD(1:40).
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3. Figure 3. DSC thermograms of TA, CS-SD, TA-CS-SD(1:10), and PM.
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4. Figure 4. FTIR spectrums of TA, CS-SD, TA-CS-SD(1:10), and PM. 5. Figure 5. SEM micrographs of (A) TA, (B) CS-SD, (C) PM, (D) TA-CS-SD(1:5), (E) TA-CS-SD(1:10), and (F) TA-CS-SD(1:20).
6. Figure 6. Degradation (A) and Swelling (B) profiles of CS-SD.
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7. Figure 7. Percent A549 cell viability (% control) treated 24 h with the different formulations: blank medium, DMSO, CS-SD dispersed in medium, TA dispersed in medium, TA dissolved in DMSO, and TA-CS-SD (1:10) dispersed in medium
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respectively (n = 5). *P < 0.05, **P < 0.01 and ***P < 0.001 compared with TA
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1. A novel tanshinone IIA solid dispersion is fabricated using chitosan as carrier for
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the first time. 2. The dissolution rate of tanshinone IIA is improved by solid dispersion approach. 3. The release mechanism of solid dispersion exhibits pH-responsive performance.
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4. The crystallinity of tanshinone IIA is decreased by solid dispersion approach, and
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the reduction of crystallinity is correlated with the content of chitosan matrix. 5. In vitro cytotoxicity test is performed to verify the bioactivity improvement of
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tanshinone IIA solid dispersion.